Chikungunya virus (CHIKV) is a mosquito-transmitted, single-stranded RNA alphavirus of the Togaviridae family that causes explosive epidemics of a severe febrile illness characterized by debilitating polyarthalgias in humans. CHIKV (NIAID Category C pathogen) caused an estimated 1.4 million new cases in India alone in 2006, and has the potential to spread globally because of the distribution and abundance of its primary mosquito vector, Aedes aegypti. During recent outbreaks, more severe forms of CHIKV infection were observed including encephalopathy and hemorrhagic fever, suggesting the emergence of more virulent strains. Currently, no specific treatment or vaccine is available. Given its global burden, the increased travel into CHIKV-endemic areas, and the worldwide spread of its vector, there is a pressing need for the development of prophylactic and therapeutic agents against CHIKV. In preliminary experiments, we have generated strongly neutralizing monoclonal antibodies (MAbs) against CHIKV that prevent infection in mice. Here, an established academic-industry collaborative partnership between the Diamond laboratory and MacroGenics will develop humanized antibody therapeutics that protect against virtually all strains of CHIKV. In Aim 1, panels of mouse MAbs against the E1 and E2 envelope proteins of CHIKV will be generated and screened functionally. The most potent neutralizing MAbs that recognize an array of genetically diverse CHIKV strains will be evaluated for protection in mice. In Aim 2, mechanistic correlates of antibody protection will be defined by characterizing the cellular stages of antibody blockade. Additionally, we will identify CHIKV recognition determinants using high-throughput yeast surface display epitope-mapping technology. In Aim 3, we will engineer humanized MAbs that block infection of CHIKV. These will be tested for neutralizing activity in cell culture and in mice. Humanized MAbs will be tested for protective efficacy, durability, and therapeutic effect in vivo. High-expressing cell lines producing the best candidates will be established. Overall, the generation of strongly protective humanized MAbs against CHIKV will foster the clinical development of immunotherapeutic agents against CHIKV infection.